Vitamin D binding protein (DBP) is an abundant serum glycoprotein synthesized by the liver which transports vitamin D and its metabolites, and binds to actin monomers to remove them from the circulation after tissue injury. DBP or DBP-related proteins of undetermined function have also been identified on the surface and/or in the cytosol of variety of cells. Recent findings from this laboratory demonstrate two size classes of DBP-related mRNAs in several non-liver rat and human tissues, suggesting further complexity in the DBP gene family and its functions. This project involves a study of the gene family encoding DBP and these DBP-related molecules using as molecular probes the rat and human DBP cDNAs, which were generated and analyzed during the first two years of this work. The specific aims are: to determine the full spectrum of tissues in which DBP-related genes are transcribed, to determine if these transcripts encode a molecule different than liver DBP and, if so, to clone and sequence cDNA clones encoding these mRNAs, to establish their encoding chromosomal loci, to determine the nature and extent of any post-translational modifications of the encoded proteins, to establish whether they bind vitamin D metabolites and/or actin, and if the DBP-related proteins are not secreted, to establish their intracellular locations. Using cDNA probes for DBP and the related transcripts, the ontogeny and tissue specificity of their expression will be studied and compared to data for the related albumin and Alpha-fetoprotein genes. The correlation between functional domains and structure in DBP and the related proteins will be studied by targeted cDNA mutagenesis and expression, followed by binding studies with 125I-actin and [3H]25(OH)D. The position of DBP in the albumin/Alpha-fetoprotein gene cluster will be established, and the structural genes encoding DBP and related proteins will be isolated. These genes will be partially characterized by DNA sequence analysis, and used for transfection experiments to further explore the tissue-specific and hormonally-regulated patterns of gene expression. The long-term objectives of this proposal include a complete biochemical characterization of this gene family in order to determine the role of DBP and proteins in normal steroid hormone action and in pathological states related to their absence, genetic variation, or abnormal functioning.